PATHOGENESIS AND TREATMENT OF APLASTIC ANEMIA

再生障碍性贫血（AA）和其他类型的骨髓衰竭具有与自身免疫病理生理一致的临床和实验室特征，具有多种刺激性抗原，包括病毒、化学物质和药物。无论其具体病因如何，大多数患者在免疫抑制治疗后血液学改善。AA的一个重要临床特征是其演变，有时在血球计数正常化数年后，演变为其他血液学疾病，如阵发性夜间血红蛋白尿（PNH），这源于造血干细胞的克隆。我们的实验室研究主要集中在AA的免疫病理生理，病毒抗原的鉴定，以及晚期克隆进化的机制。病因学研究继续关注于AA型肝炎后综合征中一种未知肝炎病毒（见Z01 HL 02319-14 HB）。目前对免疫系统在骨髓抑制中的作用的研究主要集中在γ -干扰素（γ - ifn）上，这是一种在体外和体内抑制造血的淋巴因子。在我们的免疫介导的AA动物模型中，先天性淋巴细胞诱导深度骨髓发育不全，这可以通过早期给药抗淋巴细胞球蛋白（ATG）、环孢素和γ - ifn单克隆抗体来消除。目前的努力是通过定期输注淋巴样细胞来产生慢性发育不全，以及确定免疫反应的特异性。我们用流式细胞术测量了循环淋巴细胞和骨髓淋巴细胞中的γ - ifn。大约2/3的严重AA患者和诊断为低细胞性骨髓增生的患者在血液T细胞中显示出这种细胞因子的存在；不到10%的康复患者的检测结果为阳性。细胞因子的存在高度预测免疫抑制治疗的反应：16/16的细胞含有γ - ifn的患者有反应，而4/12的血液缺乏γ - ifn的患者有反应；治疗后IFN含量下降，IL-4含量升高。细胞内干扰素的复发预示着复发。骨髓细胞内IFN可能与反应有更好的相关性。表面γ - ifn的测量允许分离活的活化T细胞，这可能有助于确定淋巴细胞的克隆性和特征，甚至识别其抗原。体外环孢素、各种雄激素制剂和生长因子联合治疗也会改变TH1/TH2平衡的细胞内细胞因子测量；干细胞因子和粒细胞集落刺激因子也可以抑制正常干细胞动员个体体内IFN的表达。在再生障碍性贫血的其他临床研究中，24名新诊断的严重再生障碍性贫血患者现已被随机分配到比较方案中；ATG组的反应率高于高剂量环磷酰胺组，但复发和细胞遗传学异常仅发生在ATG组。对于儿童，我们对标准方案进行了修改，缩短了环孢素治疗的持续时间，为了避免取消ATG耐受性，开始环孢素治疗的时间推迟，并添加了新的免疫抑制剂霉酚酸酯，所有这些都是为了降低高复发率。在实验室，晚期克隆性疾病，无论是PNH和骨髓发育不良，使用敏感的流式细胞术和荧光原位杂交测定。大约20%的AA患者和更大比例的低细胞性骨髓增生患者存在PNH克隆的证据。我们的结果与PNH代表免疫介导的骨髓衰竭逃逸机制的假设一致，并且PNH的发展需要两个步骤。另外，糖基磷酸肌醇连接蛋白可能参与初始抗原刺激。-自身免疫，T细胞，骨髓衰竭，骨髓增生，免疫抑制，干扰素-人类受试者

Aplastic anemia (AA) and other types of bone marrow failure have clinical and laboratory features consistent with an autoimmune pathophysiology, with a diversity of inciting antigens, including viruses, chemicals, and drugs. Whatever its specific etiology, a majority of patients respond with hematologic improvement after immunosuppressive therapies. One important clinical feature of AA is its evolution, sometimes years after normalization of blood counts, to other hematologic diseases such as paroxysmal nocturnal hemoglobinuria (PNH), which derive from clones of hematopoietic stem cells. Our laboratory studies have focused on the immune pathophysiology of AA, identification of a viral antigen, and the mechanism of late clonal evolution. Studies of etiology have continued to focus on an unknown hepatitis virus in the post-hepatitis AA syndrome (see Z01 HL 02319-14 HB). Current studies of the immune systems role in bone marrow suppression have focused on gamma-interferon (gamma-IFN), a lymphokine that inhibits hematopoiesis in vitro and in vivo. In our animal model for immune-mediated AA, congenic lymphocytes induce profound marrow aplasia, which can be abrogated by early administration of anti- lymphocyte globulin (ATG), cyclosporine, and monoclonal antibody to gamma-IFN. Current efforts are directed to producing chronic aplasia by periodic infusions of lymphoid cells, as well as determination of the specificity of the immune response. We have measured gamma-IFN in circulating and marrow lymphocytes using flow cytometry. About 2/3 of patients with severe AA and also with the diagnosis of hypocellular myelodysplasia shown the presence of this cytokine in blood T cells; less than 10% of recovered patients scored positive. The presence of the cytokine was highly predictive for response to immunosuppressive therapies: 16/16 patients whose cells contained gamma-IFN responded, compared to 4/12 patients whose blood lacked gamma-IFN; IFN declined after treatment, while IL-4 content increased. Recurrence of IFN intracellularly predicted relapse. Marrow intracellular IFN may be even better correlated with response. Measurement of surface gamma-IFN allows isolation of viable activated T cells, which may be useful in determining lymphoid clonality and characterizing and even identifying their antigens. Intracellular cytokine measurements indicating TH1/TH2 balance are also altered by in vitro treatment with cyclosporine, various androgen preparations, and growth factor combinations; stem cell factor and granulocyte colony stimulating factor also depress IFN expression in vivo in normal individuals undergoing stem cell mobilization. In other clinical studies in aplastic anemia, 24 patients with newly diagnosed severe aplastic anemia have now been randomized in a comparative protocol; the response rate has been higher for ATG than for high dose cyclophosphamide, but frank relapses and cytogenetic abnormalities have occurred only in the ATG arm. For children, our standard protocol has been modified to shorten the duration of cyclosporine therapy, begin cyclosporine is begun later in order to avoid abrogation of ATG tolerization, and the new immunosuppressive agent mycophenolate mofetil has been added, all in an effort to decrease the high relapse rate. In the laboratory, late clonal disease, both PNH and myelodysplasia, using sensitive flow cytometric and fluorescent in situ hybridization assays. Approximately 20% of patients with AA and probably a larger proportion of cases of hypocellular myelodysplasia present with evidence of a PNH clone. Our results are consistent with the hypothesis that PNH represents an escape mechanism in immune-mediated bone marrow failure, and that two steps are required for the development of PNH. Alternatively, a glycosylphosphoinositol- linked protein may be involved in the initial antigenic stimulation. - autoimmunity, T cells, bone marrow failure, myelodsyplasia, immunosuppression, interferon - Human Subjects